香菇C91-3转录本Unigene 24277基因克隆表达及其生物学活性的研究

摘要

通过对香菇C91-3转录本Unigene 24277基因的生物信息学分析，克隆表达含RCC1结构域的Unigene 24277基因，并研究其抗肿瘤活性。从香菇C91-3菌丝体中提取总RNA，反转录合成cDNA，并利用Rapid Ampli-fication of cDNA Ends（ RACE）技术获得基因全长。NCBI 数据库分析提示其含有 RCC1结构域。PCR 扩增RCC1结构域，将其克隆产物与pET-32a（+）载体连接，热转化至E. coil Rosetta-gami（DE3）中诱导表达，纯化、复性后，通过MTT法研究其抗肿瘤活性。结果显示原核表达载体构建成功，重组蛋白成功诱导表达，并初步证明了重组蛋白具有抑制肿瘤细胞增殖活性的功能，为后续抗肿瘤机制的探究奠定了基础。%Xanggu mushroom( Lentinula edodes)C91-3 gene transcripts Unigene 24277 was cloned and expressed in Unigene 24277 gene of RCC1 structure domain through bioinformatic analysis and studied its anti-tumor activity. Total RNA was extracted from L. edodes C91-3 mycelium,cDNA was synthesized by reverse transcription and obtained the full-length gene by Rapid Amplification of cDNA Ends( RACE)technology. The NCBI database analysis indicated that it contains RCC1 structure domain. Amplified the RCC1 structure domain by PCR and connected the cloned prod-ucts with pET-32a( +)vector,thermal conversion into E. coli Rosetta-gami(DE3)to induce expression,after puri-fication and refold,its anti-tumor activity was studied by MTT method. The results showed that prokaryotic expression vector was constructed successfully and successfully induced the expression of recombinant proteins. It was preliminary proved that the recombinant protein possessed the function of inhibiting tumor cell proliferation,and laid a foundation for the follow-up study on the exploration of the mechanism of anti-tumor.